The present invention relates to plants which exhibit improved resistance to certain pathogenic agents which are sensitive to stilbenes, and relates, more specifically, to a set of constructs which combine a plant promoter which can be induced by a biotic stress, which stress is engendered, in particular, by the said pathogens, with (a) gene(s) encoding a stilbene synthetase.
A large part of the world harvest of cultivated plants is regularly destroyed by parasites and pathogens. Among the possible options for decreasing or preventing the attack by these parasites on cultivated plants, chemical control (plant protection treatments) is the method which is most used. Nevertheless, the application of chemical products is not without consequences for the environment and sometimes presents technological problems as, for example, the appearance of new resistant pathogenic strains or, in the field of oenology, the difficulties which can arise during fermentations (the use of inhibitors of sterol biosynthesis can block yeast growth at the end of fermentation) or the presence of chemical products, such as procymidone, an anti-Botrytis product, which are sometimes found in wine.
The control method which consists in improving the resistance of cultivated plants to the diseases which are caused by these pathogens has been envisaged as a way of overcoming the drawbacks associated with chemical control. It is possible, for example, in a first approach, to achieve this improvement by the sexual route, i.e. using classical genetics, by hybridizing the plants whose resistance is to be improved with tolerant varieties. Nevertheless, this approach is not always feasible (tolerant natural variety not known) or is not permitted by legislation such as, for example, in viticulture as a result of French legislation on Appellations d""Origine Controlxc3xa9e (A.O.C.) (registered designations of origin) which limits the grapevine varieties which are to be used for a given appellation (designation).
In a second approach, it is possible, using the modern techniques of cell and molecular biology, to integrate, into the genome of the plant, one or more homologous or heterologous genes which make it possible to overexpress or express a molecule of interest, which is of protein nature, in order to increase the production of a metabolite, or a metabolic pathway, or to open a new biosynthetic pathway or to synthesize a novel molecule for example for increasing the opening of a new biosynthetic pathway, for example increasing the resistance of the plant by reinforcing its defence mechanisms with regard to the pathogens in question.
There are several different defence mechanisms of this type in plants. Some can be regarded as being passive and are linked to the physicochemical characteristics of the cells, the epidermal tissues and/or the organs of the plant. Others belong to the dynamics of gene/gene interactions (plant resistance genes and pathogen avirulence genes, mechanisms of host/pathogen interactions). While these interactions can lead to the development of a hypersensitivity reaction (rapid death of the cells of the plant around the point of infection in order to block colonization of the plant by the microorganism), they can also lead to the synthesis and accumulation of a whole series of compounds. Of these, some can be parietal constituents which are involved in the formation of a xe2x80x9cphysicalxe2x80x9d barrier around the point of infection (callose, lignin, hydroxyproline-rich protein: HRGP, etc.), and other compounds can be molecules having antimicrobial functions which are more or less well defined (phytoalexins, pathogen-associated proteins: PR proteins (pathogenesis-related proteins), etc.). The molecules of the phytoalexin type which are synthesized and accumulated by plants during, for example, host/pathogen interactions include, in particular the stilbenes, which are toxic, in particular for microorganisms. The term stilbene designates a group of chemical substances which possess the trans-diphenyl-1,2-ethylene skeleton as the common basal structure, with resveratrol and pinosylvine being among the simplest. This basal skeleton is synthesized in plants by a stilbene synthetase or related enzymes from substrates such as malonyl-CoA, cinnamoyl-CoA or coumaroyl-Coa, which are substances which are present in all plants (flavonoid precursors). Genes for stilbene synthetase or related enzymes have been isolated, sequenced and cloned, in particular from groundnut, orchid and grapevine. Using these genes, it has been possible to transform plants such as potato, lucerne or tobacco, with these plants then exhibiting greater resistance than untransformed plants to pathogen attack (EP-309862; EP-648839; MELCHIOR, F. et al., Arch. Biochem. Biophys. 1991, 288, 2, 552-557; WIESE, W. et al., Plant Mol. Biol. 1994, 26,2,667-677; HAIN, R. et al., Nature 1993, 361, 153-156).
The expression or overexpression of these molecules having antimicrobial functions can provide plants with a xe2x80x9cnaturalxe2x80x9d resistance in response to stresses, in particular stresses of the microbial type. However, constitutive overexpression of this type of protein necessarily has disadvantages for the plant (energy cost, slowing down of growth, etc.) (FISCHER, R. et al., The Plant Journal 1997, 11, 3, 489-498).
On the other hand, in some plants, such as grapevine or herbaceous plants, stilbenes are only found in some healthy tissues and at very low concentrations. Conversely following an infection or a lesion, these stilbenes increase strongly at the infected or damaged site, since the stilbene synthetase genes are inducible under conditions of biotic or abiotic stress (for example wounds, ultraviolet rays, etc.).
Nevertheless, this regulation is rarely present in plants of agricultural interest or, when it is present, it can be insufficiently effective. For example, studies on phytoalexin synthesis in the grapevine have demonstrated that the only healthy tissue in which stilbenes, including resveratrol, are present is healthy wood tissue. Stilbene is found to be present in the tissues of the grape berry when, on the one hand, the berry has been subjected to a stress such as attack by a pathogen (Botrytis cinerea for grey mould or Plasmopora viticola for grape downy mildew) and, on the other hand, only during the period up to the incipient ripening of the young fruit. By contrast, the concentration of the stilbene decreases strongly from incipient ripening to maturation. However, damage due, for example, to Botrytis is rarely encountered during the period up to incipient ripening but rather during the period close to maturation of the berry. For this reason, expression of the stilbene synthetase gene has to be controlled with strong promoters which escape the natural regulation of the gene and which should be inducible, in particular by the stress itself. The present invention specifically relates to a promoter of this nature.
The present invention relates to nucleic acids which comprise the sequence of the promoter for a lucerne PR protein linked to at least one sequence of a gene encoding a stilbene synthetase.
The invention relates, in particular, to nucleic acids according to the invention, characterized in that the promoter for a lucerne PR protein is a promoter which can be induced in plants, in a tissue-specific manner or not, by a biotic or abiotic stress.
The invention also relates to nucleic acids according to the invention, characterized in that the sequence of the promoter for a lucerne PR protein is selected from the group comprising:
a) the IND S1 sequence,
b) any sequence corresponding to a fragment of the IND S1 sequence and having a promoter sequence effect in plants.
The sequences of the promoter for a lucerne PR protein are preferred which exhibit at least 80% homology with the IND S1 sequence. Those sequences are particularly preferred which exhibit at least 90% or 95% homology with the said sequence.
The sequences of promoters for lucerne PR proteins according to the invention were obtained from regulatory sequences of genes for PR proteins by taking advantage of the incompatibility response (hypersensitivity reaction, HR) obtained in the host/parasite relationship between lucerne (Medicago sativa) and Pseudomonas syringae pv pisi for the purpose of isolating the regulatory sequences of genes which are responsible for this reaction.
When Pseudomonas attacks lucerne, the occurrence of a plant reaction is observed in the region adjacent to the necrosis caused by the bacterial infection.
Plant material was therefore removed following the bacterial attack in order to construct a cDNA library from the messenger RNAs which were produced in the regions adjacent to the necrosis. Amplification by polymerase chain reaction (PCR), using synthetic polynucleotides corresponding to motifs which are conserved in leguminous PR protein genes, enabled a radioactive probe to be obtained which was then used to select transcripts in the cDNA library. One of these (cDNA-PR7) was adopted since, after sequencing, it exhibited good homology with equivalent genes encoding PR proteins and known from other plants (cf. FIGS. 1 and 1a, depicting the general scheme of the method for isolating the promoter).
Analysis showed that it corresponded to a gene encoding a class 10 PR protein according to the VAN LOON (1994) classification. This gene was therefore designated Ms PR10-1 (Medicago sativa PR class 10 protein, clone 1). The isolated and cloned cDNA PR7 made it possible to obtain two probes due to the presence of an internal BamHI site (B in FIG. 1). These probes, EB and BE (E corresponding to the EcoRI site in FIG. 1) named respectively 5xe2x80x2 and 3xe2x80x2, were used to screen a lucerne genomic library. Among the clones obtained which were recognised by the 5xe2x80x2 and 3xe2x80x2 probes, one of them, C15, was selected and sequenced (6.1 Kb). It itself also possesses, logically, a BamHI site which made it possible to obtain two novel EB and BE fragments of 2.4 kb and 3.7 kb, named respectively E-B(g) and B-E(g), g indicating the genomic nature of the fragments obtained (see FIG. 1a). Fragment E-B(g), which is located in 5xe2x80x2 of the C15 clone, and which comprises the promoter and a portion of its Ms PR10-1 gene coding sequence, was inserted into the EcoRI and BamHI sites of the bluescript plasmid. The plasmid was linearized by means of a PstI site which is located upstream of BamHI in fragment E-B(g). A deletion from 3xe2x80x2 to 5xe2x80x2 was carried n out on this fragment until the IND S1 promoter sequence was obtained (FIG. 3). A blunt end ligation made it possible to reposition another BamHI site, internal to the C15 clone, at the end of the promoter sequence of the Ms PR10-1 gene. Under these conditions, 13 nucleotides of the coding sequence of the Ms PR10-1 gene, located upstream of this BamHI site which is internal to the C15 clone, were thus integrated into the IND S1 promoter sequence: the whole can be isolated with an EcoRI/BamHI digestion (see Example 1).
In the description, PMs PR 10-1 is also intended to mean any nucleic acid fragment of the IND S1 sequence with a promoter effect in plants and the IND S1 sequence with 13 nucleotides of the coding sequence of the Ms PR10-1 gene as described below.
The invention also relates to nucleic acids according to the invention, characterized in that the sequence of a gene encoding a stilbene synthetase, whether homologous or heterologous, is selected from the genes isolated from groundnut, orchid, grapevine and pine genomes (EP-309 862, EP-464 461).
Of the said nucleic acids, preference is given to the nucleic acids which encode a grapevine stilbene synthetase, in particular those described in the article by HAIN, R. et al., Natrue 1993. 361, 153-156 and in that by WIESE, W. et al., Plant Mol. Biol., 1994, 26, 2, 667-677; the nucleic acid corresponding to the sequence vst1 of the said articles is that which is most preferred.
The nucleic acids enabling the stilbene synthetase gene(s) to be expressed will naturally be able to include, in particular, apart from the said gene(s), polyadenylation sequences at the 3xe2x80x2 end of the coding strand and enhancer sequences from the said gene or from a different gene.
Naturally, the nucleic acid sequences will have to be adapted in order to ensure that the gene is actually read in the correct reading frame with the promoter and it will obviously be possible to foresee using, if necessary, several promoters of the same type as well as several enhancer sequences.
It is also possible to use the nucleic acids according to the present invention to express several stilbene synthetase genes, either arranged in tandem or carried by different expression systems.
The nucleic acids according to the invention can be used to create expression systems in plants, which systems can be inducible and/or constitutive depending on the tissues or organs of the plant which are transformed (cf. Examples 2, 3 and 4).
The present invention therefore also relates to systems for expressing at least one stilbene synthetase gene in plants, characterized in that they comprise at least one nucleic acid according to the invention. Of the systems according to the invention, preference is given to transformation vectors, particularly transformation vectors of the plasmid type.
Advantageously, the said transformation vectors are characterized in that they can be transferred into Agrobacterium strains.
The stilbene synthetase genes which are able to be expressed by the nucleic acids according to the present invention are placed under the control of the PMs PR10-1 promoter for the purpose of activating, in plants, mechanisms of resistance to pathogens which are sensitive to stilbenes, in particular to resveratrol, to pinosyl grapevine or to their glycosylated derivatives such as picein or to oligomers such as the viniferins. Parasites of this nature which are sensitive to stilbenes and which may be mentioned are Botrytis cinerea, Plasmopora viticola, Eutypa lata, etc.
Preference is given to those expression systems according to the invention which are characterized in that they can be induced in plants by a biotic or abiotic stress.
From the said biotic stresses according to the a invention preference is given in particular to biotic stresses which are engendered by the attack of a parasite which is sensitive to stilbenes, such as a virus, a bacterium, a yeast, a fungus, in particular Botrytis cinerea or Plasmopora viticola. 
From the said abiotic stresses according to the invention, preference is given in particular to abiotic stresses engendered by a mechanical wound such as that caused in particular by an insect or by a physical phenomenon such as wind or frost.
The present invention also relates to plant cells which are transformed with a system or a vector according to the invention. Advantageously, the said plant cells are grapevine cells.
The present invention also relates to processes for transforming plant cells using a microbiological method, including the systems or vectors according to the present invention.
The invention furthermore relates to processes for obtaining plants which express one (or several) stilbene synthetase gene(s), characterized in that cells of the said plants are transformed using a system or a vector according to the invention, the cells expressing the said gene(s) are selected and a plant is regenerated from these cells.
The most frequently used transformation methods which may be mentioned are, in particular, the methods which employ Agrobacterium, whether this be Agrobacterium tumefaciens or Agrobacterium rhizogenes, biolistic or any other techniques (electroporation, etc.).
These methods are known (REAM, W., 1989; NEGRETIU, I. and GHARTI-CHHETRI, G. B., 1991; CASSE-DELBART, F., 1996; STANFORD, J. C., 1990) and will not be described again in detail.
The technology, which in particular makes use of plasmid systems, enables a first transformation of a competent bacterial strain, in general E. coli, to be effected, which transformation enables the structure of the plasmids to be cloned and monitored. The strain is then used to transfer the recombinant plasmids into agrobacterial strains, which will then be used to transform the plant cells.
The plants comprising an expression system or cells according to the invention are part of the invention.
The plants which are obtained by implementing the processes according to the invention are also part of the invention.
Finally, the invention relates to plants according to the invention, characterized in that the plants are plants of agricultural interest, in particular grapevine plants.
Other characteristics and advantages of the constructs and the processes according to the present invention will be evident from the examples which follow.